We have learned an enormous amount in the past three years bout the mechanism and biology of one of the major components of the human genome, the L1 (LINE) retrotransposon. This remarkable element is likely responsible, directly or indirectly, for about one third of our genome by weight; its reverse transcriptase ORF (ORF2) is the most abundant ORF in the human genome. Recent gains in understanding how this element works result from technical breakthroughs. We and our colleagues developed and exploited a new assay for L1 retrotransposition in human tissue culture cells and also, we identified and characterized a critical new functional domain of the L1 element, the endonuclease. We will use multiple systems, including yeast genetics, biochemistry, bioinformatics and human tissue culture cells to analyze the molecular mechanisms by which the L1 element replicates and inserts itself into new sites. We have developed several in vitro assays for the functions of the ORF2 protein, including target-primed reverse transcription (TPRT) and a potentially novel reverse transcription reaction that does not require addition of a primer. We will also explore several proposed mechanisms by which the potentially deleterious process of L1 retrotransposition may be regulated in cells, including DNA methylation and cis-action of L1 proteins. We will evaluate possible biological relationships Between L1 and Alu sequences as well as between retrotransposons and telomeres (the latter in collaboration with the Greider laboratory). Finally, we will analyze L1 insertion sites and L1 distribution patterns on human chromosomes, taking advantage of the enormous volume of human sequence data that are already available and will soon grow to include the entire genome.